Method for Raman chemical imaging of endogenous chemicals to reveal tissue lesion boundaries in tissue

ABSTRACT

Apparatus and methods for spatially resolved Raman detection of molecules indicative of the borders of lesions with normal tissue are disclosed. A region of biological tissue was illuminated with monochromatic light. A Raman shifted light signal is detected from endogenous molecules in the region, the molecules being spatially organized in a localized first area of the region. These molecules are indicative of a border between normal tissue and a lesion. The Raman shifted light signal is then spatially resolved in at least one direction.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority pursuant to 35 U.S.C. 119(e) to U.S.Provisional Application No. 60/301,708 filed Jun. 28, 2001 which isincorporated herein by reference in its entirety including incorporatedmaterial.

FIELD OF THE INVENTION

The field of the invention is the field of tissue evaluation and, moreparticularly, the field of tissue evaluation using optical detection oflight which has been Raman shifted in frequency.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of death in the United States andover 1.2 million people are diagnosed with this disease annually. Canceris significant, not only in lives lost, but also in the $107 billioncost to the United States economy in 2000 according to the NationalInstitutes of Health. It is widely recognized among the cancer researchcommunity, that there is a need to develop new tools to characterizenormal, precancerous, cancerous, and metastatic cells and tissues at amolecular level. These tools are needed to help expand our understandingof the biological basis of cancers. Molecular analysis of tissue changesin cancer improve the quality and effectiveness of cancer detection anddiagnosis strategies. The knowledge gained through such molecularanalyses helps identify new targets for therapeutic and preventativeagents.

Diagnosis of cancer is the first critical step to cancer treatment.Included in the diagnosis is the type and grade of cancer and the stageof progression. This information drives treatment selection. When canceris suspected, a patient will have the tumor removed or biopsied and sentfor histopathology analyses. Conventional handling involves the tissueundergoing fixation, staining with dyes, mounting and then examinationunder a microscope for analysis. Typically, the time taken to preparethe specimen is of the order of one day. The pathologist will view thesample and classify the tissue as malignant or benign based on theshape, color and other cell and tissue characteristics. The result ofthis manual analysis depends on the choice of stain, the quality of thetissue processing and staining, and ultimately on the quality ofeducation, experience and expertise of the specific pathologist.

Early definitive detection and classification of cancerous growths isoften crucial to successful treatment of this disease. Currently,several biopsy techniques are used as diagnostic methods after cancerouslesions are identified. In the case of breast cancer, lesions aretypically identified with mammography or self breast exam. The mostreliable method of diagnosis is examination of macroscopic-sizedlesions. Macroanalysis is performed in conjunction with microscopicevaluation of paraffin-embedded biopsied tissue which is thin-sectionedto reveal microscale morphology.

The detection and diagnosis of cancer is typically accomplished throughthe use of optical microscopy. A tissue biopsy is obtained from apatient and that tissue is sectioned and stained. The prepared tissue isthen analyzed by a trained pathologist who can differentiate betweennormal, malignant and benign tissue based on tissue morphology. Becauseof the tissue preparation required, this process is relatively slow.Moreover, the differentiation made by the pathologist is based on subtlemorphological differences between normal, malignant and benign tissuebased on tissue morphology. For this reason, there is a need for animaging device that can rapidly and quantitively diagnose malignant andbenign tissue.

Alternatives to traditional surgical biopsy include fine needleaspiration cytology and needle biopsy. These non-surgical techniques arebecoming more prevalent as breast cancer diagnostic techniques becausethey are less invasive than biopsy techniques that harvest relativelylarge tissue masses. Fine needle aspiration cytology has the advantageof being a rapid, minimally invasive, non-surgical technique thatretrieves isolated cells that are often adequate for evaluation ofdisease state. However, in fine needle biopsies intact breast tissuemorphology is disrupted often leaving only cellular structure foranalysis which is often less revealing of disease state. In contrast,needle biopsies use a much larger gauge needle which retrieve intacttissue samples that are better suited to morphology analysis. However,needle biopsies necessitate an outpatient surgical procedure and theresulting needle core sample must be embedded or frozen prior toanalysis.

A variety of “optical biopsy” techniques have potential as non-invasive,highly sensitive approaches that will augment, or even be alternativesto current diagnostic methods for early detection of breast cancer.“Optical biopsies” employ optical spectroscopy to non-invasively probesuspect tissue regions in situ, without extensive sample preparation.Information is provided by the resultant spectroscopically uniquesignatures that may allow differentiation of normal and abnormaltissues. Despite years of research and development, two techniques thathave not realized their potential are:

-   -   (1) fluorescence optical biopsies, which fails due to the        nonspecific nature of tissue autofluorescence; and    -   (2) near-infrared optical diagnostics, in particular        non-invasive glucose sensing, which fails due to interference        from tissue major components, including predominantly water.

In contrast to other techniques, Raman spectroscopy holds promise as anoptical biopsy technique that is anticipated to be broadly applicablefor characterization of a variety of cancerous disease states. A numberof researchers have shown that Raman spectroscopy of masses of cells hasutility in differentiating normal vs. malignant tissue anddifferentiating normal vs. benign tissue. In general, the Raman spectraof malignant and benign tissues show an increase in protein content anda decrease in lipid content versus normal breast tissue, demonstratingthat cancer disease states impact the chemistry of the tissue.

However, Raman spectroscopy has not been able to differentiate benignvs. malignant tissues due to the spectral similarities of these tissuetypes. In addition, Raman spectroscopy of breast tissue samples requireslarge numbers of cell populations. If only a small portion of the cellsare cancerous, as in the early stages of lesion development, then Ramanspectroscopy of a large number of such cells will be insensitive to thedisease. It would be advantageous to have a technique capable of thespatial sensitivity needed for discrimination of cancerous from normalcells in early stage breast cancer diagnosis.

Chemical imaging based on optical spectroscopy, in particular Ramanspectroscopy, provides the clinician with important information.Chemical imaging simultaneously provides image information on the size,shape and distribution (the image morphology) of molecular chemicalspecies present within the sample. By utilizing molecular-specificimaging, based on chemical imaging, the trained clinician can make adetermination on the disease-state of a tissue or cellular sample basedon recognizable changes in morphology without the need for samplestaining or modification.

Apparatus for Raman Chemical Imaging (RCI) has been described by theinventors in U.S. Pat. No. 6,002,476, and in co-pending U.S.Non-provisional application 09/619,371 filed Jul. 19, 2000 which claimsbenefit of U.S. Provisional application 60/144,518 filed Jul. 19, 1999.The above identified U.S. patents, patent applications, and publicationsare hereby incorporated by reference.

OBJECTS OF THE INVENTION

It is an object of the invention to produce apparatus and methods usingRaman shifted light for diagnosis of lesions in tissue. It is an objectof the invention to produce apparatus and methods for diagnosis oftissue samples excised from a patient. It is an object of the inventionto produce apparatus and methods for in vivo diagnosis of tissue. It isan object of the invention to produce apparatus and methods for findinga lesion in vivo in tissue. It is an object of the invention to produceapparatus and methods for determining the borders of lesions in vivo andin tissue samples excised from a patient. It is an object of theinvention to produce apparatus and methods for spatially resolving Ramanshifted light from tissue in vivo and in tissue samples. It is an objectof the invention to produce apparatus and methods for imaging a lesionwith light which has been Raman shifted.

SUMMARY OF THE INVENTION

Raman chemical imaging is used to differentiate between normal tissueand benign and malignant lesions. In particular, Raman chemical imagingis shown to be sensitive to calcified tissue and to carotenoidmolecules. Carotenoid molecules are concentrated at the border of alesion, and can be used to indicate the borders of the lesion. Spatiallyresolved Raman signals indicate lesions and borders of lesions. Othermolecular species which may be indicative of border regions are noted.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A. Brightfield reflectance microscope image of a calcified lesionfrom a five micron thick frozen section biopsy.

FIG. 1B. Magnified region indicated from FIG. 1A.

FIG. 1C. Raman Chemical Image (RCI) of the spatial distribution ofcalcification (calcium hydroxyapetite) and background (microscope slide)

FIG. 2A. Brightfield reflectance microscope image of a region ofcalcified lesion as indicated in FIG. 1A.

FIG. 2B. Polarized light image of the region of interest indicated inFIG. 1A.

FIG. 2C. Raman Chemical Image (RCI) indicating calcified tissue.

FIG. 2D. Raman spectral data from two regions of interest indicated inFIG. 2C showing the different chemical composition of these regions.

FIG. 3A. Brightfield reflectance microscope image of a 5 micron thinsection of human breast tissue biopsy sample showing a lesion and theadjacent tissue.

FIG. 3B. Magnified region of interest indicated in FIG. 3A.

FIG. 3C. Raman Chemical Image (RCI) of the endogenous caroteniod(beta-carotene) that shows the border of the lesion.

FIG. 3D. Raman spectra obtained with a tunable filter for two circledregions indicated in FIG. 3C.

FIG. 4. Amount of carotenoid as determined from its spectral signalalong the diagonal A–A′ of the view shown in FIG. 1C.

FIG. 5. Preferred embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Raman Spectroscopy

When light interacts with matter, a portion of the incident photons arescattered in all directions. A small fraction of the scattered radiationdiffers in frequency (wavelength) from the illuminating light. If theincident light is monochromatic (single wavelength) as it is when usinga laser source or other sufficiently monochromatic light source, thescattered light which differs in frequency may be distinguished from thelight scattered which has the same frequency as the incident light.Furthermore, frequencies of the scattered light are unique to themolecular or crystal species present. This phenomenon is known as theRaman effect.

In Raman spectroscopy, energy levels of molecules are probed bymonitoring the frequency shifts present in scattered light. A typicalexperiment consists of a monochromatic source (usually a laser) that isdirected at a sample. Several phenomena then occur including Ramanscattering which is monitored using instrumentation such as aspectrometer and a charge-coupled device (CCD). Similar to an infraredspectrum, a Raman spectrum reveals the molecular composition ofmaterials, including the specific functional groups present in organicand inorganic molecules and specific vibrations in crystals. Ramanspectrum analysis is useful because each resonance exhibits acharacteristic ‘fingerprint’ spectrum, subject to various selectionrules. Peak shape, peak position and the adherence to selection rulescan also be used to determine molecular conformation information(crystalline phase, degree of order, strain, grain size, etc.). Unlikeinfrared spectroscopy, a single Raman spectrometer can be applied to themolecular characterization of organic and inorganic materialssimultaneously. Other advantages of Raman over traditional infraredspectroscopy include the ability to analyze aqueous phase materials andthe ability to analyze materials with little or no sample preparation.Deterrents to using Raman spectroscopy as opposed to infraredspectroscopy include the relatively weak nature of the Raman phenomenonand interferences due to fluorescence. In the past several years, anumber of key technologies have been introduced into wide use that haveenabled scientists to largely overcome the problems inherent to Ramanspectroscopy. These technologies include high efficiency solid statelasers, efficient laser rejection filters, and silicon charge coupleddevice (CCD) detectors.

In Raman spectroscopy instruments, a linear CCD array is typicallypositioned at the exit focal plane of single stage, low f number Ramanmonochromators for efficient collection of dispersive Raman spectra. Themonochromator disperses the Raman shifted light, and the CCD arraytypically produces a signal which is proportional to the intensity ofthe Raman signal vs wavelength.

Raman Chemical Imaging (RCI)

In many respects, Raman chemical imaging is an extension of Ramanspectroscopy. Raman chemical imaging combines Raman spectroscopy anddigital imaging for the molecular-specific analysis of materials. Muchof the imaging performed since the development of the first Ramanmicroprobes has involved spatial scanning of samples beneath Ramanmicroprobes in order to construct Raman “maps” of surfaces.Historically, Raman imaging systems have been built using this so calledflying spot (“point-scanning”) approach, where a laser beam is focusedto a spot and is scanned over the object field, or likewise a linescanning approach, where the laser spot is broadened in one directionby, for example, a cylindrical lens, and the two dimensional imageformed on a CCD array has one spatial dimension and one wavelengthdimension. Raman chemical imaging techniques have only recently achieveda degree of technological maturity that allows the simultaneouscollection of high-resolution (spectral and spatial) data. Advancementsin imaging spectrometer technology and their incorporation intomicroscopes that employ CCDs, holographic optics, lasers, and fiberoptics have allowed Raman chemical imaging to become a practicaltechnique for material analysis.

Imaging spectrometers include Fabry Perot angle rotated or cavity tunedliquid crystal (LC) dielectric filters, acousto-optic tunable filters,and other LC tunable filters (LCTF) such as Lyot Filters and variants ofLyot filters such as Solc filters and the most preferred filter, anEvan's split element liquid crystal tunable filter, which is describedin the March (1999) issue of Analytical Chemistry on page 175A. Otherpreferred wavelength filtering means comprise polarization-independentimaging interferometers such as Michelson, Sagnac, Twynam-Green, andMach-Zehnder interferometers.

References describing the above identified techniques that can be usedto obtain chemical images include:

-   Fiber Array Filters (FAST)—M. P. Nelson, M. L. Myrick, Appl.    Spectroscopy 53, 751–759, (1999);-   Dielectric Interference filters—D Batchelder, C Cheng, W Muller, B    Smith, Makromol Chem Macromol. Symp 46, 171, (1991);-   AOTF—P. J. Treado, I. W. Levin, E. N. Lewis, Appl. Spectrosc. 46,    1211–1216, (1992);-   Lyot—B. Lyot, C. R. Acad. Sci. 197:1593. (1933);-   Fabry Perot—K. A. Christainsen, N. L. Bradley, M. D. Morris, R. V.    Morrison, Appl. Spectrosc. 49, 120–1125, (1995);-   Solc filter—A. Yariv & P. Yeh, Optical Waves in Crystals, (Wiley    N.Y., 1984);-   Michelson Interferometer—Sybil P. Parker, Optics Source Book,    (McGraw-Hill, N.Y., 1988 p143);-   Sagnac Interferometer—S. Spielman, K. Fesler, C. B. Eom, T. H.    Geballe, M. Fejer and A Kapitulnik, Phys. Rev. Lett., 65, 123    (1990); and-   Twyman-Green Interferometer—M. Born and E. Wolf, Principles of    Optics: Electromagnetic Theory of Propogation of Light, 6th Ed,    (Pergamon Press, Oxford, 1980) pp 302–305.-   Mach-Zehnder—James D. Ingle, Jr., and Stanley R Crouch,    Spectrochemical Analysis, (Prentice Hall, Engelwood, N.J., 1988), p    83.

Raman chemical imaging is a versatile technique that is well suited tothe analysis of complex heterogeneous materials. In a typical Ramanchemical imaging experiment, a specimen is illuminated withmonochromatic light, and the Raman scattered light is filtered by animaging spectrometer which passes only a single wavelength range. TheRaman scattered light may then be used to form an image of the specimen.A spectrum is generated corresponding to millions of spatial locationsat the sample surface by tuning an imaging spectrometer over a range ofwavelengths and collecting images intermittently. Changing the selectedpassband (wavelength) of the imaging spectrometer to another appropriatewavelength causes a different material to become visible. A series ofsuch images can then uniquely identify constituent materials, andcomputer analysis of the image is used to produce a composite imagehighlighting the information desired. Although Raman chemical imaging ispredominately a surface technique, depth-related information can also beobtained by using different excitation wavelengths or by capturingchemical images at incremental planes of focus. Contrast is generated inthe images based on the relative amounts of Raman scatter or otheroptical phenomena such as luminescence that is generated by thedifferent species located throughout the sample. Since a spectrum isgenerated for each pixel location, chemometric analysis tools such ascorrelation analysis, Principle Component Analysis (PCA) and factorrotation, including Multivariate Curve Resolution (MCR) can be appliedto the image data to extract pertinent information otherwise missed byordinary univariate measures. A spatial resolving power of approximately250 nm has been demonstrated for Raman chemical imaging using visiblelaser wavelengths. This is almost two orders of magnitude better thaninfrared imaging which is typically limited to 20 microns due todiffraction. In addition, image definition (based on the total number ofimaging pixels) can be very high for Raman chemical imaging because ofthe use of high pixel density detectors (often 1 million plus detectorelements).

Applications of Raman chemical imaging range from the analysis ofpolymer blends, defect status analysis in semiconductor materials,inclusions in human breast tissue and the characterization of corrosionsamples. RCI provides a potential solution for obtaining bothqualitative and quantitative image information about molecularcomposition and morphology of breast lesions allowing a more accuratemedical diagnosis than traditional imaging methods.

Breast Tissue Results

Raman spectra can potentially reveal a wealth of information aboutmolecular properties of tissues. RCI compounds this information byallowing variations in these properties throughout the tissue to beprobed. FIG. 1 shows RCI data on a calcified lesion. The tissue wasexcised from the patient, and frozen. A five micron thick section wassliced from the tissue and prepared on a microscope slide for imaging ina microscope. FIG. 1A shows a brightfield reflectance image of a portionof the frozen sectioned biopsy which is then magnified in FIG. 1B. Thebrightfield image reveals light and dark regions resulting fromdifferences in refractive indices. These images, however, provide noinsight into the molecular makeup of the tissues at hand. A Ramanchemical image is shown in FIG. 1C and reveals the distribution ofcalcium hydroxyapatite based on its Raman response. FIG. 2A and FIG. 2Bshow dramatic differences in the optical microscopic image that dependon the polarization of the light. However, the Raman chemical image inFIG. 2C is unique in that it is derived from the distinct spectral Ramanshown in FIG. 2D. The Raman spectra in FIG. 2D shows the spectral“fingerprints” associated with the calcium hydroxyapatite and thebackground, (the glass microscope slide) respectively. Such Ramanspectra are the basis that allow a Raman Chemical image to be created.This ability to characterize calcifications is a critical issue in thediagnosis of breast carcinoma as calcification is a major element inmammographic evaluation and early cancer detection, and is critical forthe diagnositic pathologist to identify. The Raman spectrum of calciumsalts and protein calcium complexes is an incompletely explored area, inlarge part because of the previous unavailability of instrumentationcapable of simultaneous high resolution spatial imaging and highwavelength resolution Raman spectrochemical analyses.

Difficulties exist when trying to use non imaging Raman spectroscopyalone to differentiate benign vs. malignant tissues due to the spectralsimilarities of these tissue types and to the spectrum of breastconditions that may mimic cancer. In addition, non imaging Ramanspectroscopy of breast tissue samples large numbers of cell populations.If only a small portion of the cells are cancerous, as in the earlystages of lesion development, then non-imaging Raman spectroscopy willbe insensitive to the disease. It is very advantageous to have atechnique capable of the spatial imaging sensitivity needed fordiscrimination of cancerous from normal cells in early stage breastcancer diagnosis.

We have developed an imaging optical biopsy approach based on Ramanchemical imaging. In comparison with non-imaging Raman spectroscopy, ourapproach has the advantage that we efficiently collect spatial resolvedRaman spectra so that morphometric analysis (characterization by sizeand shape) can be performed in conjunction with Raman spectral analysis.The additional morphology information is anticipated to add a criticalcomponent to the analysis of disease states, in part because it buildsupon traditional cancer histopathology methods and could therefore bereadily adopted by pathologists. FIG. 3A shows a brightfield image of a5 □m thin section human breast tissue biopsy sample viewed under themicroscope. An enlarged section of the lesion is indicated and magnifiedin FIG. 3B to show the border or interface between a tumor and normaltissue, where both cancerous and normal cells are visible. The Ramanchemical image of a carotenoid molecule, β-carotene, shown in FIG. 3Creveals the location of the tumor and carotenoid molecules. Note thatthe carotenoid molecules are associated with the border between thelesion and the normal tissue. The LCTF-generated Raman spectra in FIG.3D shows the spectral “fingerprints” associated with the tumor and thetypical normal tissue, respectively. The ability to see this boundarywith an inherent chemical within human tissue is a unique finding withpotential biological and clinical significance relating to the objectivescreening and characterization of tumor margins.

FIG. 4 shows the results of a scan of the carotenoid signal along thediagonal A–A′, ie along a line perpendicular to the tumor normal tissueboundary of FIG. 3C.

It is very important to know where the tumor margins are, and to know ifthe tumor has infiltrated beyond the a well defined boundary and intonormal tissue. Detection of molecules indicative of the boundary is ofgreat importance. The nutritional literature supports the idea thatcarotenoids are protective from cancer. It is surmised but not proven bythe inventors that such protective molecules accumulate in the borderregion between a lesion and normal tissue, and act to prevent the lesionfrom growing. Other molecules suggested by the nutritional literature inrelation to breast cancer are indoles, sulforaphanes, and flavonoids.Proteoglycans molecules have been noted to be associated with prostatecancer. With the Raman chemical imaging, the position of thesemolecules, and molecules which will be identified in the future, may beclearly imaged and used to show the extent and the stage of growth ofthe cancer or other lesion.

The cancerous cells shown in the lesion in FIGS. 3B and 3C are alsodifferentiated from adjacent cells in the Raman image based on molecularcompositional variations (lipid vs. protein content primarily) and canalso be used to create a Raman image of the diseased tissue. As aresult, the images are molecule-specific and more specific than imagesderived from stains. Because the Raman scattering of the tissues isintrinsic to the tissues, stains are not required and the technique issuitable for in vivo use. The Raman images are collected in only severalseconds using laser power density that does not modify the tissuesamples.

An in vivo embodiment of the invention for examining a breast 50 orother non-arterial soft tissue for a lesion 51 is shown in FIG. 5. Anendoscope or other instrument 52 is used to introduce light carried byan optical fiber 53 from a monochromatic light source 54. A dichroicmirror 55 and lens 56 are shown schematically for introducing the lightinto the fiber 53. Raman light from the breast is carried from thebreast tissue back through the lens 56 and mirror 55, through a filter57 to a detector 58. The signal from the detector 58 is analyzed by acomputer system 59 and displayed on a monitor 60.

Filter 57 is most preferably a Evan's split element liquid crystaltunable filter, which is controlled by computer 59.

The endoscope 52 is preferably an imaging endoscope or fiberscope, wherelight is conducted from the breast tissue to the detector 58 in acoherent manner through a large plurality of optical fibers. A series oftwo dimensional images is preferably taken as a function of depth intothe tissue and of the Raman shifted wavelength.

Results of a preferable embodiment of the invention is shown by aninsert in FIG. 5, where the signal shown is a signal of a moleculeindicative of a border region between the breast 50 or other nonarterial soft tissue and the lesion 51. The spatially resolved signal ofcalcified tissue or of, for example, carotenoid molecules, is shown inthe insert as a function of depth into the breast as the needle carryingthe optical fiber is moved into the breast. The signal is showndisplayed on the display device 60. In this embodiment, a much finerneedle is used than the needle carrying an imaging endoscope. In thefine needle embodiment, the location of the lesion may be moreaccurately determined, so that fine needle aspiration cytology and/orneedle core biopsy may be performed. In the fine needle embodiment, thefilter 57 may be a normal spectrometer or a liquid crystal tunablefilter, preferably of the Evan's split element type.

Raman chemical imaging also has demonstrated utility for thequantitative assessment of lesions in breast tissues. However, there isa need to make systematic strides in the development of a RCI opticalbiopsy. RCI of animal breast tissue models have been analyzed, as wellas studies of human cancer lesions. Other lesions besides benign andmalignant tumors, such as pockets of infection and inflamation will showup in the Raman chemical images. Several data treatment methods havebeen utilized to analyze the Raman image data which include bandratioing, band shift analysis, and classical linear least squaresanalysis. Comparisons have been made between the various processingapproaches that address the utility of RCI for breast tissue componentdiscrimination.

Applications

There is a great need for an instrument that can provide: real timedetection with accuracy, decreased patient discomfort and recovery,minimal cosmetic defect of the breast, minimal distortion of the breasttissue that might make interpretation of future mammograms difficult andmost importantly provide the patient with rapid feedback on hercondition.

The user base for an instrument suitable for objective assessment ofbreast lesions of will consist of medical research laboratories,University and non-affiliated hospitals, and private clinics.

On another level, the customer or end-user is the patient that requiresthe procedure be completed to determine the disease state of her breasttissues. At this level the numbers are as follows: more than 1,000,000biopsies were conducted in 1997; the growth rate for biopsies is almost20% annually as clinicians struggle with how to determine the diseasestate of tissue early enough to prevent radical measures; the typical“customer” is a woman over the age of 40 that should be having annualbreast exams by a clinician; and the number of potential customers isapproximately 57 million (women between ages of 40 and 85).

The benefits to the target users of RCI systems will be substantial.Configured in an endoscopic version of the technology, RCI can beemployed for “real-time” breast tissue evaluation tool that iscompatible with and complementary to existing, mature clinicalapproaches (namely, needle core biopsies). When performed in combinationthe effectiveness of breast cancer diagnosis will likely be enhanced.Benefits will include, but are not limited to, the following:

-   -   Real-time evaluation of suspicious lesions sites identified        through self-breast exam and/or mammography that are made        accessible via needle core biopsy.    -   Immediate feedback to the clinician as to the severity of the        clinical situation. Results can be communicated to the patient        by the physician shortly after completion of Raman biopsy.    -   Potential information on prognostic indicators of disease such        as growth rate through quantitative evaluation of cellular        nucleic acid composition and proliferation associated peptides.    -   Minimal patient discomfort.    -   Minimal to no cosmetic defect of the breast.    -   Reduced exposure to ionizing radiation (x-rays).    -   Specific applications of a RCI system for evaluating breast        lesions will include the following:    -   Discrimination of malignant vs. benign tumors    -   Spatial distribution of carotenoids in tissues    -   Spatial distribution of calcified tissue    -   Spatial distribution of proteins, lipids and carbohydrates in        tissues

Advantages Over Currently Available Technology

Traditional approaches to identification of breast lesions includeself-breast exam and x-ray mammography. These techniques are effectiveas initial screening techniques, especially when performed incombination. Unfortunately, mammography is associated with a high falsepositive rate, resulting in 3–7 patients being biopsied for everypatient cancer diagnosed. Although many mammographic abnormalities aredefinitely benign, and others are obviously malignant, there are manylesions in which the diagnosis cannot be made with certainty based onthe mammographic appearance alone. To verify the disease-state of adetected lesion, tissue must be sampled for pathologic examination. Thismay be done with fine needle aspirates, core biopsies, or excisionalbiopsies. These samples are then prepared, stained, and inspected by atrained pathologist. This process can take several days to completebefore the patient is informed of the outcome. Raman chemical imagingtechnology has the potential to assist diagnosis of the disease state ofbreast lesions in real-time.

Currently, several biopsy techniques are used as diagnostic methodsafter breast lumps are identified, typically with mammography,ultrasound, or breast examination. The most reliable method of diagnosisis examination of macroscopic-sized lesions. Macroanalysis is performedin conjunction with microscopic evaluation of paraffin-embedded biopsiedtissue which is thin-sectioned to reveal microscale morphology.

Alternatives to traditional surgical biopsy include fine needleaspiration cytology and needle core biopsy. These non-surgicaltechniques are becoming more prevalent as breast cancer diagnostictechniques because they are less invasive than conventional biopsytechniques that involve surgical incision. Fine needle aspirationcytology has the advantage of being a rapid, minimally invasive,non-surgical technique that retrieves cytologic material that is oftenadequate for evaluation of disease state. However, in fine needlebiopsies breast tissue histologic features are minimal, leaving onlycytologic features for analysis of disease state. In contrast, needlebiopsies use a much larger gauge needle which retrieve tissue samplesthat are better suited to morphology analysis. However, needle biopsiesnecessitate an outpatient surgical procedure and the resulting needlecore sample must be fixed, embedded and processed prior to analysis.

State-of-the-Art Raman Chemical Imaging Techniques

Several Raman chemical imaging technologies have evolved that competewith widefield tunable filter-based RCI. These techniques include pointscanning RCI, line imaging RCI, RCI using interference filters,Fourier-transform interferometry, Hadamard-transform scanning and FASTtechnology.

Point scanning involves taking a complete spectrum for a single X,Yposition of a sample followed by raster-scanning the sample for theremaining X,Y positions. This method offers advantages of high spectralresolution and full spectral resolution, but lacks high image definitioncapabilities and is extremely time consuming. Line imaging involvescollecting data from vertical sections of the sample characterized by asingle value of X and all values of Y, followed by subsequent scanningin the X direction. This method has the nearly the same advantages anddisadvantages as the point scanning approach, but can be done morerapidly. Field curvature artifacts are a consequence of line imagingwhich degrade image quality. The use of single or multiple interferencefilters can be used to produce a wavelength specific image(s). Thismethod is rapid, cheap and produces high definition images, but lacksspectral resolution and is susceptible to image artifacts.Fourier-transform interferometers use a mechanically driveninterferometer with a CCD-based detection system. Interferograms areimaged with the CCD for subsequent spectral interpretation for each stepof the interferometer. This method boasts good spatial resolution butsuffers from poor spectral resolution (˜100 cm⁻¹). Hadamard transformchemical imaging techniques couple Hadamard mask spatial multiplexingwith CCD-based detection to obtain two spatial and one spectraldimension of data. This method offers S/N advantages for low-light levelapplications such as Raman spectroscopy in addition to sub-nanometerspectral resolution. However, the technique suffers from fair spatialresolution and poor temporal resolution since the latter involvesscanning through numerous coding masks. Fiber array spectral translators(FAST) use a two dimensional arrangement of Raman collection fiberswhich are drawn into a one dimensional distal array at the opposite end.The one dimensional fiber stack is coupled to an imaging spectrograph.Software then extracts the spectral/spatial information which isembedded in a single CCD image frame. FAST is capable of acquiringthousands of full spectral range, position-specific Raman spectra andwavenumber-specific Raman chemical images in seconds. However, the imagedefinition of FAST is limited by the number of pixels in any onedirection of the CCD chip used in the detection system (typically nobetter than 45×45 (˜2048) imaging elements).

The ideal chemical imaging system for characterization would providefast acquisition times (seconds), high spatial resolution (sub-micron)and good spectral resolution (<0.2 nm). To date, systems equipped withliquid crystal tunable filters are the only RCI system that meets theserequirements.

Other Spectroscopy-Based Imaging Methods

Spectroscopic technologies that compete with Raman such as fluorescenceand infrared (IR) spectroscopy are not of great concern based on theresolution needed to see molecules on the order of 250 microns. Althoughfluorescence has showed some promise, it suffers from low specificitywithout the use of invasive dyes or stains that require FDA approval. IRspectroscopy cannot compete due to the difficulty with water absorptionin the IR. Tissues do not image well because of their aqueous nature.Systems equipped with LCTFs surpass any dispersive grating oracousto-optic tunable filter (AOTF) technology on the market. Thespectral bandpass capability of the LCTF is 8 cm⁻¹ allowing for the mosteffective means to obtain image detail.

Traditional Biomedical Imaging Methods

Traditionally, biomedical imaging has been divided into capturing imagesof live tissue (in vivo) at relatively low resolution (from 10 to 1000microns) and capturing images of excised tissue at high resolution. Invivo imaging is usually performed using non-optical modalities such asmagnetic resonance imaging, ultrasound, or x-ray tomography, whichassess the general shape and appearance of tissue in its native state;however, this approach does not provide the cellular resolutionnecessary to analyze cell types and tissue morphology. To image tissueat high resolution using conventional optical or electron microscopes,one had to slice the tissue into thin sections, otherwise the tissueabove and below the layer of interest will produce out-of-focusreflections that seriously degrade image contrast. Confocal techniquesaddress this to some extent. Excising, fixing and staining thin tissuesections is however static, is time-consuming and by the very nature ofthe process involves tissues which have been rendered non viable.

A RCI system will produce quantitative digital images of the lesiontissue that will be recognizable to the clinician who makesdisease-state determinations, in large part, based on the visualappearance of images. The appearance of suspect tissue, when viewed bythe naked eye if lesions are large enough, or via x-ray mammography, orvia magnetic resonance imaging (MRI) provides important clues to thestate of the tissue. After years of training, clinicians can basediagnosis on these subtle visual clues. Despite the best efforts ofhighly skilled professionals, early stage disease-state determination isa difficult problem. By aiding the pathologist with an image that mapsthe distribution of certain molecular species, the large number ofsubjective determinations of disease state in breast tissue biopsies canbe greatly reduced.

Although we have described certain present preferred embodiments of ourmethod for objective evaluation of breast tissue using Raman imagingspectroscopy, it should be distinctly understood that our invention isnot limited thereto, but may include equivalent methods. It is furtherto be distinctly understood that the present invention is not limited tothe evaluation of breast tissue and applies to the evaluation of alltissue. Obviously, many modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that, within the scope of the appended claims, theinvention may be practiced otherwise than as specifically described.Publications, patents, and patent applications noted herein are herebyincluded by reference.

1. A method for detecting a border between normal tissue and a lesionwhich comprises a) illuminating a region of biological tissue withmonochromatic light; b) detecting Raman shifted light from endogenousmolecules in the region with a Raman chemical imaginingspectrophotometer, wherein the molecules are indicative of a borderbetween normal tissue and a lesion; and c) spatially resolving the Ramanshifted light signal in at least one direction to produce a Ramanchemical image indicative of a border between the normal tissue and thelesion in said at least one direction.
 2. The method of claim 1, whereina two-dimensional chemical image of the region is produced.
 3. Themethod of claim 2, where the Raman shifted light from the region passesthrough a FAST fiber array spectral translator.
 4. The method of claim2, where the Raman shifted light from the region passes through a FabryPerot tunable filter.
 5. The method of claim 2, where the Raman shiftedlight from the region passes through an acousto-optic tunable filter. 6.The method of claim 2, where the Raman shifted light from the regionpasses through a liquid crystal tunable filter.
 7. The method of claim6, where the Raman shifted light from the region passes through a Lyotfilter.
 8. The method of claim 7, where the Raman shifted light from theregion passes through an Evan's split element liquid crystal tunablefilter.
 9. The method of claim 7, where the Raman shifted light from theregion passes through a Solc filter.
 10. The method of claim 2, wherethe Raman shifted light from the region passes through apolarization-independent imaging interferometer.
 11. The method of claim10, where the Raman shifted light from the region passes through aMichelson interferometer.
 12. The method of claim 10, where the Ramanshifted light from the region passes through a Sagnac interferometer.13. The method of claim 10, where the Raman shifted light from theregion passes through a Twynam-Green interferometer.
 14. The method ofclaim 10, where the Raman shifted light from the region passes through aMach-Zehnder interferometer.
 15. The method of claim 2, where themolecules are chosen form the group consisting of indoles,sulforaphanes, carotenoids, proteoglycans and flavonoids.
 16. The methodof claim 15, where the molecules are carotenoid molecules.
 17. Themethod of claim 15, where the molecules are indole molecules.
 18. Themethod of claim 15, where the molecules are sulforaphane molecules. 19.The method of claim 15, where the molecules are flavonoid molecules. 20.The method of claim 15, where the molecules are proteoglycan molecules.21. The method of claim 2, where the region is prepared forillumination, in a step previous to step a), by excision of the tissueand by placing a specimen prepared from the tissue in position forillumination and imaging, wherein the tissue is not treated withstaining agents.
 22. The method of claim 2, where the region is preparedfor illumination, in a step previous to step a), by excision of thetissue and by placing a specimen prepared from the tissue in positionfor illumination and imaging, wherein the tissue is treated withstaining agents.
 23. The method of claim 1, wherein said region isilluminated in vivo with monochromatic light introduced via anendoscope.
 24. The method of claim 23, wherein a two-dimensionalchemical image of the region is produced.
 25. The method of claim 24,where the Raman shifted light from the region passes through a FASTfiber array spectral translator.
 26. The method of claim 24, where theRaman shifted light from the region passes through a Fabry Perot tunablefilter.
 27. The method of claim 24, where the Raman shifted light fromthe region passes through an acousto-optic tunable filter.
 28. Themethod of claim 24, where the Raman shifted light from the region passesthrough a liquid crystal tunable filter.
 29. The method of claim 28,where the Raman shifted light from the region passes through a Lyotfilter.
 30. The method of claim 29, where the Raman shifted light fromthe region passes through an Evan's split element liquid crystal tunablefilter.
 31. The method of claim 24, where the Raman shifted light fromthe region passes through a polarization-independent imaginginterferormeter.
 32. The method of claim 31, where the Raman shiftedlight from the region passes through a Michelson interferometor.
 33. Themethod of claim 31, where the Raman shifted light from the region passesthrough a Sagnac interferometer.
 34. The method of claim 31, where theRaman shifted light from the region passes through a Twynam-Greeninterferometer.
 35. The method of claim 31, where the Raman shiftedlight from the region passes through a Mach-Zehnder interferometer. 36.The method of claim 23, where the molecules are chosen form the groupconsisting of indoles, sulforaphanes, carotenoids, proteoglycans andflavonoids.
 37. The method of claim 36, where the molecules arecarotenoid molecules.
 38. The method of claim 23, where a non-imagingendoscope is moved through the tissue in vivo, and the Raman shiftedlight signal is spatially resolved as the endoscope is moved through thetissue.
 39. The method of claim 23, where the Raman shifted light fromthe region passes through an Evan's split element liquid crystal tunablefilter.
 40. The method of claim 23, wherein the endoscope is movedthrough the tissue to obtain a Raman chemical image from more then oneregion of said tissue.
 41. The method of claim 40, wherein atwo-dimensional chemical image is produced for each region of saidtissue.
 42. The method of claim 41, wherein said two-dimensionalchemical images represent a series of images taken as a function ofdepth of the tissue.
 43. The method of claim 41, wherein said multipleregions are in series as a function of depth of the tissue.
 44. A methodof performing an optical biopsy which comprises a) illuminating multipleregions of biological tissue in viva with monochromatic light introducedvia an endoscope; b) detecting Raman shifted light from endogenousmolecules in each region with a Raman chemical imagingspectrophotometer, wherein the molecules are indicative of a borderbetween normal tissue and a lesion; c) spatially resolving the Ramanshifted light signal in two dimensions for each region to produce aRaman chemical image of said tissue in two dimensions; and d)determining the location of any lesion in said tissue.
 45. A biopsymethod which comprises a) obtaining the Raman chemical image ofbiological tissue according to the method of claim 44; and b) performinga needle core biopsy of tissue in or around said lesion.
 46. The methodof claim 45, wherein said multiple regions are in series as a functionof depth of the tissue.
 47. The method of claim 46 which furthercomprises examining said biopsied tissue for signs of cancer.